Process of producing reference bactericidal endpoint (RBE) limonene

ABSTRACT

A process for producing a stable, bactericidal and fungicidal composition which consists essentially of bubbling air or oxygen into limonene, to produce an oxidized limonene having a rapid bactericidal activity as evidenced by the capability of killing substantially all of the bacteria in a suitable culture medium inoculated with 10 6  colony forming units of Staphylococcus aureus ATCC 25923 per ml. of said broth, containing not more than 0.06 ml of said oxidized limonene per ml. of said broth and incubated at 37° C. in air for 60 minutes.

This is a divisional of copending application Ser. No. 07/531,570 filedon Jun. 1, 1990, which is now U.S. Pat. No. 5,153,229.

TECHNICAL FIELD

The object of this invention is to show an economical process ofproducing oxidized limonene which has a potent, rapidly actingantimicrobial activity that persists when stored, and which is superiorin antimicrobial activity to that of auto-oxidized limonene.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

During the study of fresh limonene as a hand cleaner, the applicantsfound that fresh limonene is an excellent solvent, but it is notbactericidal. Conversely, limonene which has undergone auto-oxidation,unexpectedy, was found to have a minimal, inconsistent antibacterialactivity which varied in potency from batch to batch. Therefore, it wasdecided to purposely oxidize limonene to try to enhance and stabilizethe antimicrobial properties of the limonene.

The applicants found that by bubbling air (or oxygen) to limonene topurposely oxidize (or oxygenate limonene which is the same) the oil,that it became bactericidal at progressively decreasing concentrationsuntil it reached a point beyond which further oxidation caused adecrease in its bactericidal activity. As the purposeful oxidation oflimonene progressed, it was noted that the physical properties of thelimonene changed as its antimicrobial activity increased. Limonene,which was purposely oxidized was found to be effective in killingbacteria, yeasts, and fungi and its antimicrobial activity persistedunchanged when the purposely oxidized limonene was stored at 2°-4° C. inthe dark, for periods exceeding one year.

The ability to produce limonene with a stable antimicrobial activity wasunexpected and is not appreciated nor delineated in the prior art. Infact it is recognized in the prior art that the addition of air (oroxygen) to terpenes produces volatile mixtures of oxides and peroxidesthat are unstable. Limonene which undergoes auto-oxidation has aninconsistent antibacterial effect that is not rapidly bactericidal. Itevaporates to a thick, non-usable gel before it becomes an effectiveantimicrobial when auto-oxidation is allowed to occur over a largesurface area of the oil. On the other hand, limonene that is purposelyoxidized becomes an excellent, stable bactericide and fungicidal that isbactericidal and fungicidal in bactericidal and fungicidalconcentrations and its activity can be demonstrated with even shortinterval application.

Thus, the object of this invention is to economically produce oxidizedlimonene: (1) with a potent, rapidly acting antimicrobial activity, (2)that will retain its antimicrobial activity when stored, and (3) that issuperior in its antimicrobial activity to the antimicrobial activity ofauto-oxidized limonene.

Limonene is a terpene with the following formula: ##STR1##

Limonene is an oil with a terpenic aroma. It is soluble in alcohol andmiscible in oil, but it is insoluble in glycerine. Limonene is usuallyderived from either a pine or citrus origin. The l-isomer is derivedfrom pine and has a characteristic pine aroma while the d-isomer isderived from citrus and has a characteristic citrus aroma. The l-isomeris also made synthetically, but the synthetic l-limonene has nocharacteristic aroma.

Commercial limonene is usually obtained from the peel of citrus fruit.Pressed liquor is obtained from the peel of any citrus fruit, but mostcommonly from oranges and grapefruit, after which it is passed through aflash evaporation at 240° F. at atmospheric conditions. Limonene iscollected from the condensation of the flash vaporization. Limonene isused commercially as a flavoring agent, is used in large quantities toproduce carvone which has a spearmint flavor, is used as a solvent, canbe used as a base for soap or perfume, is used in the manufacture ofrubber, and is useful as a penetrating oil.

Limonene undergoes auto-oxidation when it is exposed to air or oxygen.After exposure to air at room temperature, limonene becomes rancid witha foul smelling odor. The temperature has to be above about 40° F. forauto-oxidation to occur while temperatures above 90° F. increase therate of evaporation. Because auto-oxidation limits the commercial valueof limonene by causing it to become rancid with a foul smelling odor,limonene is usually stored in 55 gallon drums that have their interiorpainted with phenolic resin base enamel especially formulated forterpenes. The drums are completely filled and capped in order to limitexposure to air (and oxygen) that causes auto-oxidation. Auto-oxidizedlimonene develops antibacterial activity during auto-oxidation but doesnot become a rapidly acting bactericide. Auto-oxidation does not producethe potent, rapidly bactericidal limonene described in this patent.

(2) Description of the Prior Art

The oxidation of limonene produces several chemical compounds that havebeen enumerated by several different investigators. For instance Bain(in U.S. Pat. Nos. 2,863,882 and 3,014,047) showed a method of producingand recovering terpenic oxidation products in which he delineated theoxidation products of limonene as: limonene-1,2-oxide,limonene-8,9-oxide, 1-menthene-9-ol, α-2,8-p-menthadiene-1-ol,β-2,8-p-menthadiene-1-ol, dihydrocarvone, β-cymenol, carvone,cis-carveol, and trans-carveol. He never studied the antimicrobialactivity of oxidized limonene nor oxidized limonene to the point that itbecame stable with a potent antimicrobial activity. In fact, becauselimonene is chemically unstable during its oxidation, Bain thought thatin order to obtain the maximum number of oxidation products fromlimonene, that he should add alkali to limonene when the peroxide valuewas between 1000 and 2000 and should cease adding air or oxygen tolimonene. He thought the peroxide value was the sole indicator of theformation of limonene oxidation products (oxides and peroxides) and thatthese products could be obtained only when the oxidation of limonene wasstopped as the peroxide value rose to its maximum. He never realizedthat the maximal activity achievable through the oxidation of limoneneis obtained before maximum peroxide formation occurs.

Blumann listed the compounds formed by the auto-oxidation of limonene inChemical Abstracts, Volume 63, 1965, on page 1819, and found minuteamounts of cis and trans-carveol, trans-p-menth-8-ene-1,2-diol, limonene1,2-epoxide, limonene 8,9-epoxide, cis and trans-p-mentha-2,8-dien-1-ol,and perillyl alcohol, but he never tried to purposely oxidize limoneneand never tried to develop limonene with a stable, rapidly bactericidalactivity.

Bardyshev studied the chemical compounds in photo-initiated,auto-oxidized, 1-limonene and found tiny amounts of the followingcompounds: carvone, carveol, trans-p-menth-8-ene-1,2,-diol,hydroperoxide, p-menthane, p-cymene, terpinolene, p-mentha-2,4(8)-diene,cyclohexene, cuminic aldehyde, dihydrocarvone, piperitone,p-mentha-2,8-dien-1-ol, pulegone, carvomenthone, PhCOMe, 4-MeC₆ H₄ COMe,cyclohexanone, perillyl alcohol, and p-CH₂ :CMeC₆ H₄ CH₂ OH as wasoutlined in Chemical Abstracts, Volume 80, 1974, page 359. Like Blumann,Bardyshev never purposely oxidized limonene and never studied theantimicrobial activity of fresh nor auto-oxidized limonene.

Zuckerman studied the effect of d-limonene on bacteria and found thatfresh limonene had no effect on bacteria. He found that when limonenebecame auto-oxidized, it developed minimal, inconsistent, weaklyinhibitory (not bactericidal) properties. He noted that, at best,auto-oxidized limonene was only weakly inhibitory in that it only"shifts the time of the lag phase of bacterial growth without anyinfluence on the logarithmic phase of bacterial growth." He noted thatauto-oxidized limonene is unstable, and loses its inhibitory effect onkeeping as was discussed in Nature 168: 517 (1951). He also recognizedthat the addition of alkali, as advocated by Bain in his method ofoxidizing limonene, destroys the bacteriostatic properties ofauto-oxidized limonene. He never recognized that limonene can bepurposely oxidized to a point that it becomes a potent, stableantimicrobial which retains its antimicrobial activity when stored. Inshort, he could not make oxidized limonene with a stable, rapidlybactericidal activity.

In U.S. Pat. No. 3,595,975 Gauvreau showed a means of producingdisinfecting compositions by combining cetyl pyridinium with terpenes toform antiseptics, but he never learned that limonene which had beenpurposely oxidized, becomes a potent, stable bactericide and fungicide.The active ingredient in his disinfecting compositions was cetylpyridinium chloride (and not the terpenes).

DISCLOSURE OF THE INVENTION

This invention relates to an economical and safe process of producingoxidized limonene that has a potent, stable antimicrobial activity whichis retained when it is stored. Its antimicrobial activity is superiorto, and distinct from, the antimicrobial activity of auto-oxidizedlimonene. In one preferred use the oxidized limonene of this inventionis a microbicide. Wherever used herein, the term "microbe" as used aloneor in derivatives such as microbicide and antimicrobial, means amicroscopic organism, such as bacteria, fungi, yeast, germs, viruses,and rickettsia.

During the purposeful oxidation of limonene it was found that oxidizedlimonene became rapidly bactericidal as oxidation progressed. When thebactericidal activity of oxidized limonene: (a) became greater than thatwhich could be accomplished by the auto-oxidation of limonene and, (b)could be demonstrated at a concentration of 0.06 or less againstStaphylococcus aureus ATCC 25923 when incubated for sixty minutes, theoxidized limonene was defined as reference bactericidal endpointlimonene (i.e. RBE limonene). RBE limonene is a potent, stable, rapidlybactericidal compound.

The production of oxidized limonene that is bactericidal at aconcentration of 0.06 or less against Staphylococcus aureus ATCC 25923when incubated for sixty minutes can be produced by continuouslybubbling air (method one) or oxygen (method two) through limonene atroom temperature for three to eight weeks. The applicants prefer tobubble air into limonene because oxygen is more expensive to use andoxygen has a great potential to explode, making it much more hazardousto handle. After limonene is oxidized and becomes a potent, stablebactericide, regardless of whether it is produced by the addition of airor oxygen, it retains its antimicrobial activity when stored, maintainsits pleasant terpenic aroma (as opposed to the rancid odor ofauto-oxidized limonene), and changes physical characteristics with anincreased viscosity and an increased specific gravity. While themajority of limonene specimens which are purposely oxidized develop apotent, rapidly bactericidal activity, occasional limonene specimens donot become bactericidal with oxidation regardless of the length of, orthe method of, oxidation. Thus, every batch of limonene which ispurposely oxidized (to produce RBE limonene) must be individually testedfor bactericidal activity. In short, the applicant's preferred method ofproducing oxidized limonene with a potent, stable bactericidal activityis to bubble air into limonene, because bubbling air through limonene isless expensive than using oxygen, and is less hazardous to use thanoxygen.

Oxidized limonene is slightly viscous and readily adheres to glass,metal, wood, cloth, rope, book covers, cement, canvas, ceramic tile,plant surfaces, skin, mucous membranes and teeth leaving a film of oil.In practice, any surface on which it is desirable to kill or preventbacterial or fungal growth, is contacted directly with bactericidal orfungicidal concentrations of limonene which has been oxidized until ithas developed a minimum bactericidal concentration of at least 0.06 orless for Staphylococcus aureus ATCC 25923 when incubated for sixtyminutes. It can be applied by swabbing, wiping, painting, washing,brushing, spraying, or any other direct application technique.Alternatively, it can be incorporated in creams, ointments, tinctures,gels, suppositories, paints, sprays, aerosols, toothpastes, solutions,emulsions, surgical soaps, mouthwashes, or antiseptics and can beapplied anywhere it is desirable to kill or prevent the growth ofbacteria, fungi, or yeasts.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples are illustrative of the best mode for carryingout the invention. They are, obviously, not to be construed aslimitative of the invention since various other embodiments can readilybe evolved in view of the teachings provided herein.

EXAMPLE 1

In Example 1, the several methods of producing purposely oxidizedlimonene that is bactericidal are demonstrated. In the first example,the process of bubbling air into limonene to produce oxidized limonenethat is bactericidal at a concentration of 0.06 or less forStaphylococcus aureus ATCC 25923 when incubated for 60 minutes is shownin Table A below. The method of producing auto-oxidized limonene isshown in Table B.

The process of producing purposely oxidized limonene and auto-oxidizedlimonene was as follows: Four gallons of fresh d-limonene (FloridaChemical Company, Inc., Lake Alfred, Fla.) were divided into one gallonportion in small-mouthed metal container. Two portions were treated atroom temperature (˜25° C.) while two were treated at 50° C. (in a waterbath). For each temperature, one portion was left open to room air(auto-oxidized) while the second portion was actively oxidized bycontinuously bubbling air through it with a Second Nature Challenger IPump aerator (purposely oxidized). Aliquots were removed from each ofthe four portions at 0, 2, 4, 6, 8, 10, 12, 14, and 16 weeks. For eachaliquot, the viscosity, specific gravity, peroxide value and theantibacterial activity against Staphylococcus aureus ATCC 25923 andEscherichia coli ATCC 25922 were determined as outlined in Tables A, B,D, and E below.

For those portions treated at room temperatures (Tables A and B), therewas a clear difference in the type and potency of the antibacterialactivity produced through the purposeful oxidation and auto-oxidation ofd-limonene. The purposely oxidized limonene (Table A) showed maximumbactericidal activity by 6 to 8 weeks of treatment. It was rapidlybactericidal (10 minutes exposure) at dilutions up to 1:6.25 for S.aureus ATCC 25923 and 1:800 for E. coli ATCC 25922. It was bactericidalat dilutions of 1:1600 for both organisms with more prolonged exposuretime (24 hours).

                                      TABLE A                                     __________________________________________________________________________    Changes in Physical Parameters, Peroxide Value, and Bactericidal Activity     of d-Limonene.sup.1                                                           During the Purposeful Oxidation of the Oil at Room Temperature.               Purposeful                                                                              Bactericidal Concentration.sup.3 For                                Oxidation.sup.2 at                                                                      S. aureus ATCC 25923 After                                                                   E. coli ATCC 25922 After                             Room Temperature                                                                        Incubation for Incubation for Peroxide                                                                           Specific                                                                           Kinematic                   in Weeks  10 Min.                                                                            60 Min.                                                                            24 Hr.                                                                             10 Min.                                                                            60 Min.                                                                            24 Hr.                                                                             Value.sup.4                                                                        Gravity.sup.5                                                                      Viscosity.sup.6             __________________________________________________________________________     0 (fresh)                                                                              >1.sup.7                                                                           >1   0.2  1    1    0.10  50.2                                                                              0.8439                                                                             1.035                        2        1    0.5  0.0025                                                                             0.24 0.02 0.0012                                                                             344.3                                                                              0.8535                                                                             1.176                        4        1    0.10 0.0006                                                                             0.06 0.0012                                                                             0.0006                                                                             327.2                                                                              0.9118                                                                             2.847                        6        0.16 0.06 0.0012                                                                             0.0025                                                                             0.0012                                                                             0.0006                                                                             353.5                                                                              0.9679                                                                             10.910                       8        0.24 0.06 0.0006                                                                             0.0012                                                                             0.0006                                                                             0.0003                                                                             334.2                                                                              1.0122                                                                             51.85                       10        1    0.10 0.0012                                                                             0.10 0.0025                                                                             0.0012                                                                             405.8                                                                              1.0420                                                                             199.7                       12        0.4  0.10 0.0025                                                                             0.005                                                                              0.0025                                                                             0.0006                                                                             389.8                                                                              1.0633                                                                             500.4                       14        0.4  0.10 0.0025                                                                             0.06 0.010                                                                              0.0012                                                                             848.3                                                                              1.0665                                                                             1,268.00                    16        0.4  0.16 0.0025                                                                             0.10 0.005                                                                              0.0006                                                                             929.9                                                                              1.0784                                                                             2,562.00                    __________________________________________________________________________     .sup.1 Florida Chemical Co., Inc., Lake Alfred, Florida                       .sup.2 Air was bubbled continuously into 1 gallon of oil in a smallmouthe     metal container with an aerator (Second Nature Challenger I Pump).            .sup.3 Bactericidal concentration shown as the highest dilution of            limonene (i.e. lowest concentration) killing at least 99.99% of the           original inoculum.                                                            .sup.4 Milliequivalents peroxide/kg sample                                    .sup.5 at 25° C.                                                       .sup.6 at 25° C. cSt                                                   .sup.7 Undiluted oil was not bactericidal for 99.99% of the original          inoculum.   In contrast, limonene that had been allowed to auto-oxidize a     room temperature (Table B) showed maximum bactericidal activity at 14 to     16 weeks of treatment, but never reached the potency of the purposely     oxidized oil. Rapid bactericidal activity (at 10 minutes) against S.     aureus ATCC 25923 was never seen with any dilution of the auto-oxidized     oil and only weakly diluted oil (1:2.5-1:10) was rapidly bactericidal for     E. coli ATCC 25922. Even with prolonged incubation (24 hours), the     auto-oxidized oil's bactericidal potency was 17 times less than that of     the purposely oxidized oil against S. aureus ATCC 25923 and four times     less against E. coli ATCC 25922. Increasing the treatment time of the oil     beyond 16 weeks resulted in an overall decrease in potency.

                                      TABLE B                                     __________________________________________________________________________    Changes in Physical Parameters, Peroxide Value, and Bactericidal Activity     of d-Limonene.sup.1                                                           During Auto-Oxidation of the Oil at Room Temperature.                                   Bactericidal Concentration.sup.3 For                                Auto-Oxidation.sup.2 at                                                                 S. aureus ATCC 25923 After                                                                   E. coli ATCC 25922 After                             Room Temperature                                                                        Incubation for Incubation for Peroxide                                                                           Specific                                                                           Kinematic                   in Weeks  10 Min.                                                                            60 Min.                                                                            24 Hr.                                                                             10 Min.                                                                            60 Min.                                                                            24 Hr.                                                                             Value.sup.4                                                                        Gravity.sup.5                                                                      Viscosity.sup.6             __________________________________________________________________________     0 (fresh)  >1.sup.7                                                                         >1   0.2  1    1    0.10  50.2                                                                              0.8439                                                                             1.035                        2          >1 >1   0.01 1    0.24 0.005                                                                              163.5                                                                              0.8440                                                                             1.066                        4          >1 >1   0.01 0.5  0.16 0.0025                                                                             199.5                                                                              0.8477                                                                             1.082                        6          >1 >1   0.01 1    0.06 0.0025                                                                             229.5                                                                              0.8486                                                                             1.090                        8           1 1    0.01 0.5  0.06 0.0012                                                                             288.2                                                                              0.8510                                                                             1.124                       10          >1 1     0.005                                                                             1    0.06 0.0012                                                                             336.9                                                                              0.8548                                                                             1.167                       12           1 0.5  0.01 0.10 0.01 0.0025                                                                             325.7                                                                              0.8570                                                                             1.198                       14           1 0.4  0.01 0.40 0.0025                                                                             0.0012                                                                             366.1                                                                              0.8587                                                                             1.247                       16           1 0.4  0.01 0.10 0.01 0.0025                                                                             393.8                                                                              0.8626                                                                             1.299                       21          >1 1     0.005                                                                             0.16 0.005                                                                              0.0025                                     __________________________________________________________________________     .sup.1 Florida Chemical Co., Inc., Lake Alfred, Florida                       .sup.2 1 gallon of oil in smallmouthed metal container left open to room      air.                                                                          .sup.3 Bactericidal concentration shown as the highest dilution of            limonene (i.e. lowest concentration) killing at least 99.99% of the           original inoculum.                                                            .sup.4 Milliequivalents peroxide/kg sample                                    .sup.5 at 25° C.                                                       .sup.6 at 25° C. cSt                                                   .sup.7 Undiluted oil was not bactericidal for 99.99% of the original          inoculum.                                                                

For the purposely oxidized limonene (Table A), neither the highestperoxide value nor kinematic viscosity corresponded to the greatestantibacterial potency. This suggested that the antibacterial activitywas not due solely to peroxides formed during oxidation nor was it dueto mere concentration of antibacterial substances resultant from theevaporation that occurred during treatment. For the auto-oxidizedlimonene, antibacterial activity followed the peroxide value andkinematic viscosity up to 16 weeks. This suggested that theantibacterial may have been due, in large part, to peroxides andconcentration of antibacterial substances during treatment. A comparisonof the antibacterial effects of the two oils when each had a similarperoxide value clearly reveals the distinct nature of the activity ofthe purposely oxidized limonene (Table C). The auto-oxidized oil was notbactericidal at any dilution for either test organism after shortexposure (10 minutes) while the purposely oxidized oil was bactericidalat a dilution of 1:4.2 for S. aureus ATCC 25923 and 1:800 for E. coliATCC 25922. Even with prolonged exposure (24 hours), the bactericidalpotency of the auto-oxidized oil was four to eight times less than thatof the purposely oxidized oil.

                                      TABLE C                                     __________________________________________________________________________    Comparison of the Antibacterial Effects of Purposely Oxidized d-Limonene      and Auto-Oxidized d-Limonene..sup.1                                                              Bactericidal Concentration For                                           Peroxide                                                                            S. aureus ATCC 25923 After Incubation                                                              E. coli ATCC 29522 After                                                      Incubation For                       d-Limonene Preparation                                                                      Value                                                                              10 Min.                                                                              60 Min. 24 Hr. 10 Min.                                                                              60 Min. 24                    __________________________________________________________________________                                                            Hr.                   Purposely Oxidized, 8 Weeks                                                                 334.2                                                                              0.24   0.06    0.0006 0.0012 0.0006  0.0003                Auto-Oxidized, 10 Weeks                                                                     336.9                                                                              >1     1       0.005  1      0.06    0.0012                __________________________________________________________________________     .sup.1 Compiled from Tables A and B                                      

Increasing the temperature of oxidation from room temperature (˜25° C.)to 50° C. did not uniformly decrease the time necessary to producepotent, antibacterial activity in the oils (Tables D and E). For thepurposely oxidized limonene, maximum bacterial activity was reachedafter only 3 weeks oxidation (Table D). This was in contrast to the 6 to8 weeks required for purposeful oxidation at room temperature (Table A).However, the rapid bacterial potency (10 minutes exposure) was 6 to 83times less for oil purposely oxidized at 50° C. in comparison to thatpurposely oxidized at room temperature. Thus, the two processes appearedto be generating oils with unique antibacterial effects. The oilpurposely oxidized at 50° C. became so viscous at 6 weeks, it could notbe tested further for antibacterial activity since it would not mix withthe broth diluent in the assay.

                                      TABLE D                                     __________________________________________________________________________    Changes in Physical Parameters, Peroxide Value, and Bactericidal Activity     of d-Limonene.sup.1                                                           During the Purposeful Oxidation of the Oil at 50° C.                              Bactericidal Concentration.sup.3 For                                          S. aureus ATCC 25923 After                                                                   E. coli ATCC 25922 After                            Purposeful Oxidation.sup.2                                                               Incubation for Incubation for Peroxide                                                                           Specific                                                                           Kinematic                  at 50° C. in Weeks                                                                10 Min.                                                                            60 Min.                                                                            24 Hr.                                                                             10 Min.                                                                            60 Min.                                                                            24 Hr.                                                                             Value.sup.4                                                                        Gravity.sup.5                                                                      Viscosity.sup.6            __________________________________________________________________________     0 (fresh) >1.sup.7                                                                           >1   0.2  1    1    0.1   50.2                                                                              0.8439                                                                             1.035                       2         1    0.16 0.0012                                                                             0.1  0.0006                                                                             0.0006                                                                             286.1                                                                              0.9549                                                                             8.203                       4         1    0.5  0.0012                                                                             0.1  0.0025                                                                             0.0006                                                                             273.2                                                                              1.0300                                                                             141.9                       6.sup.8   1    0.4  0.01 0.01 0.005                                                                              0.0025                                                                             307.9                                                                              1.0670                                                                             1293                        8                                       226.2                                                                              1.0784                                                                             3149                       10                                       241.9                                                                              1.0863                                                                             10,526                     __________________________________________________________________________     .sup.1 Florida Chemical Co., Inc., Lake Alfred, Florida                       .sup.2 Air was bubbled continuously into 1 gallon of oil in a smallmouthe     metal container with an aerator (Second Nature Challenger I Pump).            .sup.3 Bactericidal concentration shown as the highest dilution of            limonene (i.e. lowest concentration) killing at least 99.99% of the           original inoculum.                                                            .sup.4 Milliequivalents peroxide/kg sample                                    .sup.5 at 25° C.                                                       .sup.6 at 25° C. cSt                                                   .sup.7 Undiluted oil was not bactericidal for 99.99% of the original          inoculum.                                                                     .sup.8 Oil too viscous to permit testing for antibacterial activity after     6 weeks and for other parameters after 10 weeks.                         

Auto-oxidation of limonene at 50° C. produced an oil with even moredistinct antibacterial activity (Table E). Fourteen weeks of treatmentwere required to maximize its rapid bacterial effects (10 minutesexposure) while two to four weeks of treatment were required to maximizeits longer interval (24 hours) killing. This was similar to the timeinterval required to maximize the rapid and longer interval bactericidaleffects of limonene auto-oxidized at room temperature (Table B).However, for the limonene auto-oxidized at 50° C., the bactericidalactivity determined after long interval exposure (24 hours) decreasedafter 4 weeks treatment when the test organism was S. aureus ATCC 25923but not when it was E. coli ATCC 25922. These results indicated thatauto-oxidation of limonene at 50° C. could not produce the samebacterial activity as purposeful oxidation of limonene at roomtemperature.

                                      TABLE E                                     __________________________________________________________________________    Changes in Physical Parameters, Peroxide Value, and Bactericidal Activity     of d-Limonene.sup.1 During Auto-                                              Oxidation of the Oil at 50° C.                                                   Bactericidal Concentration.sup.3 For                                          S. aureus ATCC 25923 After                                                                   E. coli ATCC 25922 After                             Auto-Oxidation.sup.2                                                                    Incubation for Incubation for Peroxide                                                                           Specific                                                                           Kinematic                   at 50° C. in Weeks                                                               10 Min.                                                                            60 Min.                                                                            24 Hr.                                                                             10 Min.                                                                            60 Min.                                                                            24 Hr.                                                                             Value.sup.4                                                                        Gravity.sup.5                                                                      Viscosity.sup.6             __________________________________________________________________________     0 (fresh)                                                                              >1.sup.7                                                                           >1   0.2  1    1    0.1   50.2                                                                              0.8439                                                                             1.035                        2        >1   1     0.0025                                                                            0.5  0.02 0.0012                                                                             308.8                                                                              0.8743                                                                             1.559                        4        >1   1     0.0025                                                                            0.4  0.02 0.0012                                                                             268.0                                                                              0.8906                                                                             2.010                        6        >1   1    0.02 0.16 0.0025                                                                             0.0025                                                                             217.5                                                                              0.9005                                                                             2.406                        8         1   1    0.01 0.2  0.0025                                                                             0.0012                                                                             189.6                                                                              0.9056                                                                             2.700                       10         1   1    0.01 0.1  0.005                                                                              0.0012                                                                             182.5                                                                              0.9135                                                                             3.168                       12        > 1  1    0.02 0.1  0.0025                                                                             0.0025                                                                             192.6                                                                              0.9201                                                                             3.748                       14         1   0.5  0.02 0.01 0.0025                                                                             0.0025                                                                             211.0                                                                              0.9282                                                                             4.544                       16         1   0.5  0.06 0.1  0.0025                                                                             0.0025                                                                             219.6                                                                              0.9363                                                                             5.516                       21        >1   1    0.02 0.06 0.005                                                                              0.005                                      __________________________________________________________________________     .sup.1 Florida Chemical Co., Inc., Lake Alfred, Florida                       .sup.2 1 gallon of oil in a smallmouthed metal container left open to roo     air.                                                                          .sup.3 Bactericidal concentration shown as the highest dilution of            limonene (i.e. lowest concentration) killing at least 99.99% of the           original inoculum.                                                            .sup.4 Milliequivalents peroxide/kg sample                                    .sup.5 at 25° C.                                                       .sup.6 at 25° C. cSt                                                   .sup.7 Undiluted oil was not bactericidal for 99.99% of the original          inoculum.                                                                

A summary of the maximal bactericidal activity obtained followingtreatment of limonene by the four processes described in the previousparagraphs is given in Table F. This activity has been separated intorapid bactericidal effects (10 minutes exposure) and longer-intervalbactericidal effects (24 hours exposure) to emphasize the unique natureof the activity produced by the purposeful oxidation of limonene at roomtemperature. From this summary it is clear that the rapid bacterialactivity can be produced only with purposeful oxidation of limonene atroom temperature. The substances in the oil responsible for the rapidbactericidal activity appear to be heat labile (since they were notgenerated at 50° C.) and are clearly not peroxides. Longer intervalbactericidal effects can be produced in limonene by a variety oftreatments, and these too appear unrelated to peroxide content of theoils. However, only purposeful oxidation of limonene at room temperatureresults in an oil with both rapid and longer interval bactericidalactivity at low concentrations.

                                      TABLE F                                     __________________________________________________________________________    Maximal Bactericidal Activity and Peroxide Value of Various Limonene          Preparations..sup.1                                                                              Rapid.sup.2 Bactericidal Activity Against                                                           Long-Interval.sup.3 Bactericidal                                              Activity Against                     Limonene Preparation                                                                             S. aureus ATCC 25923                                                                      E. coli ATCC 25922                                                                      S. aureus ATCC                                                                            E. coli ATCC             __________________________________________________________________________                                                         25922                    A.                                                                              Maximal Bactericidal Activity.sup.4                                           Purposely Oxidized, Room Temperature                                                           0.16  (6).sup.6                                                                           0.0025                                                                             (6)  0.0012                                                                              (6)   0.0006                                                                             (6)                   Purposely Oxidized, 50° C.                                                              1     (2)   0.1  (2)  0.0012                                                                              (2)   0.0006                                                                             (2)                   Auto-Oxidized, Room Temperature                                                                1     (16)  0.1  (16) 0.01  (2)   0.0025                                                                             (4)                   Auto-Oxidized, 50° C.                                                                   1     (14)  0.01 (14) 0.0025                                                                              (2)   0.0012                                                                             (2)                 B Peroxide Value.sup.5 of Oils with Maximal                                     Bactericidal Activity                                                         Purposely Oxidized, Room                                                                       353.5       353.5     353.5       353.5                      Temperature                                                                   Purposely Oxidized, 50° C.                                                              286.1       286.1     286.1       286.1                      Auto-Oxidized, Room Temperature                                                                393.8       393.8     163.5       199.5                      Auto-Oxidized, 50° C.                                                                   211.0       211.0     308.8       308.8                    __________________________________________________________________________     .sup.1 Data from Tables A, B, D, and E                                        .sup.2 Measured after 10 min. exposure of the test organism to the oil        .sup.3 Measured after 24 hr. exposure of the test organism to the oil         .sup.4 Shown as highest dilution of oil killing 99.99% of bacterial           inoculum                                                                      .sup.5 Milliequivalents peroxide/kg sample                                    .sup.6 Number in parentheses indicates weeks of treatment of the oil.    

The appearances of the oils following six weeks of the four treatmentregimens are quite different. For purposely oxidized limonene, the oiltreated at 50° C. was darker yellow and more viscous than that treatedat room temperature. For auto-oxidized, the oil treated at roomtemperature was essentially unchanged in its appearance from untreated(fresh) limonene. Limonene auto-oxidized at 50° C. was similar inappearance to that purposely oxidized at room temperature.

From the data presented in Tables A, B, D, and E, a single point wasselected as a reference point to indicate that any future oxidizedlimonene was "fully oxidized". The reference bactericidal endpoint (RBE)selected was a bactericidal activity at a dilution of at least 1:16.7(0.06) when tested against S. aureus ATCC 25923 for 60 minutes exposure.This end point was selected because it (a) was found to be highlyreproducible and required a greater dilution of oil than any 10 minuteexposure endpoint, and (b) was an indicator for the presence of a rapidbactericidal effect against S. aureus and E. coli, and (c) was notobtained with any of the treatments of d-limonene except purposefuloxidation at room temperature. Thus, hereafter, all oxidized limoneneachieving this reference bacterial endpoint is referred to as RBElimonene.

The standard assay used to test the bactericidal activity againstStaphylococcus aureus ATCC 25923 or Escherichia coli ATCC 25922 inTables A, B, C, D, E, F, H, I, & J was as follows: various volumes ofthe desired limonene were added to Mueller-Hinton broth, mixedvigorously (Vortex Genie Mixer, Scientific Products, Evanston, Ill.),inoculated with 10⁶ colony forming units (CFU)/ml of Staphylococcusaureus ATCC 25923 or Escherichia coli ATCC 25922, remixed and incubatedat 37° C. in air. In varying the volume of the desired limonene tobroth, concentrations of 1 (undiluted) to 0.01 (1:100 dilution) could betested in a final volume of one to five ml (Table G). Higher dilutionsof limonene (up to 1:51,200) were tested by preparing serial two-folddilutions from the 0.01 dilution (i.e. equal volumes oflimonene-containing broth and broth were mixed serially). At varioustime intervals, each tube was mixed vigorously and a 0.01 aliquotremoved and subcultured on 5% sheep blood agar plates to determine theapproximate number of viable CFU/ml in each tube. Results of theseassays were expressed as the highest dilution of limonene with nodetectable viable colonies on subculture i.e. the bactericidalconcentration. This represented the lowest concentration killing 99.99percent of the original inoculum.

                  TABLE G                                                         ______________________________________                                        Dilutions of limonene used in fixed volume assays                             Dilutions Tested                                                                             Limonene Concentration                                         ______________________________________                                        Undiluted      1                                                              1:2            0.5                                                            1:2.5          0.4                                                            1:3.3          0.3                                                            1:4.2          0.24                                                           1:5            0.2                                                            1:6.25         0.16                                                           1:10           0.1                                                            1:6.25         0.06                                                           1:50           0.02                                                           1:100          0.01                                                           ______________________________________                                    

To ascertain if a more potent bactericide could be produced throughauto-oxidation of d-limonene when the process occurred over a largesurface area of the oil, the following experiment was performed: Fivehundred milliliters of fresh d-limonene (Intercit Inc., Safety Harbor,Fla.) was equally divided into 250 ml portions. One portion was placedinto a 500 ml Erlenmeyer flask while the second portion was placed in a500 ml beaker. The beaker was left open, exposed to air at roomtemperature (auto-oxidized), while the sample in the flask was purposelyoxidized at room temperature by continuously bubbling air through itwith a Second Nature Challenger I Pump aerator (purposely oxidizedlimonene). An aliquot of 5 ml was removed from each (beaker or flask) atone, two, and three weeks. The viscosity, specific gravity, peroxidevalue, and the antibacterial activity against S. aureus ATCC 25923 ofeach aliquot was determined as previously outlined. In this experiment,auto-oxidation could take place over a larger surface area of the oilthan in the prior experiment (performed in the small-mouthed container).Purposeful oxidation was still performed in a small-mouthed container inthis experiment to prevent any ancillary auto-oxidation.

As shown in Table H, auto-oxidation of d-limonene at room temperatureover a large surface area of the oil, did not produce a highlybactericidal oil. The characteristics of the oil found at three weekswere most similar to those of limonene auto-oxidized over a smallsurface area after six weeks (Table B). However, oxidation over a largesurface area could not be continued for prolonged periods becauseevaporation of the oil led to a product too viscous to examine forbactericidal activity. Thus, auto-oxidation of limonene over a largesurface area did not produce a rapidly bactericidal oil and evaporationprecluded long-term oxidation under these conditions.

                                      TABLE H                                     __________________________________________________________________________    Changes in the physical parameters, peroxide value, and                       bactericidal activity of d-limonene.sup.1 during auto-oxidation of            the oil over a large surface area.                                                           Bactericidal Concentration.sup.2                               Auto-oxidation at                                                                            After Incubation for                                                                         Peroxide.sup.3                                                                     Specific.sup.4                             Room Temperature in Weeks                                                                    10 Min                                                                             60 Min                                                                             24 Hrs                                                                             Value                                                                              Gravity                                                                            Viscosity.sup.5                       __________________________________________________________________________    0 (Fresh)      .sup. >1.sup.6                                                                     >1   >1    68.7                                                                              0.8432                                                                             1.024                                 1              >1   >1   >1   131.2                                                                              0.8438                                                                             1.036                                 2              >1   >1   >1   170.5                                                                              0.8434                                                                             1.052                                 3              >1   >1   0.01 231.3                                                                              0.8454                                                                             1.071                                 __________________________________________________________________________     .sup.1 250 ml of fresh limonene (Intercit Inc., Safety Harbor, Fla. lot       509801) in an open beaker at room temperature                                 .sup.2 Bactericidal concentration shown as the highest dilution (i.e.         lowest concentration) killing at least 99.99% of the original inoculum of     S. aureus ATCC 25923                                                          .sup.3 Millequiv. peroxide/kg sample                                          .sup.4 at 25° C.                                                       .sup.5 cSt at 25°  C.                                                  .sup.6 Undiluted oil was not bactericidal                                

The bactericidal activity of purposely oxidized limonene in thisexperiment is shown in Table I. As was seen before, a rapidlybactericidal oil was produced by this process. The source of the oil wascompletely different than that shown in Table A, and the referenceendpoint (i.e. bactericidal at a concentration of 0.06 or less for S.aureus ATCC 25923) was reached sooner than the previous experiment.These results suggested that different lots of limonene may requiredifferent treatment times for purposeful oxidation to reach the definedreference endpoint.

                                      TABLE I                                     __________________________________________________________________________    Changes in the physical parameters, peroxide value, and bactericidal          activity of d-limonene.sup.1 during purposeful oxidation of the oil.                        Bactericidal Concentration.sup.3                                Purposeful oxidation at                                                                     After Incubation for                                                                         Peroxide.sup.4                                                                     Specific.sup.5                              Room Temperature in Weeks.sup.2                                                             10 Min                                                                             60 Min                                                                             24 Hrs                                                                             Value                                                                              Gravity                                                                            Viscosity.sup.6                        __________________________________________________________________________    0 (Fresh)     .sup. >1.sup.7                                                                     >1   >1    68.7                                                                              0.8432                                                                             1.024                                  1             0.1  0.1  0.1  131.2                                                                              0.8438                                                                             1.036                                  2             0.1  0.1  0.0025                                                                             170.5                                                                              0.8434                                                                             1.052                                  3             0.1  0.1  0.00125                                                                            231.3                                                                              0.8454                                                                             1.071                                  __________________________________________________________________________     .sup.1 Intercit, Inc., Safety Harbor, Florida, lot 509801                     .sup.2 Air bubbled continuously into 250 ml of limonene in a 500 ml           Erlenmeyer flask with an aerator (Second Nature Challenger I Pump) at roo     temperature.                                                                  .sup.3 Bactericidal concentration shown as the highest dilution (i.e.         lowest concentration) killing at least 99.99% of the original inoculum of     S. aureus ATCC 25923.                                                         .sup.4 millequiv. peroxide/kg sample                                          .sup.5 at 25° C.                                                       .sup.6 cSt at 25° C.                                                   .sup.7 Undiluted oil was not bactericidal                                

The ability of purposeful oxidation of limonene to produce a rapidlybactericidal oil was determined using limonene from various sources. Asshown in Table J, not all samples of limonene became bactericidal withpurposeful oxidation at room temperature. Two samples from Aldrich didnot acquire bactericidal activity even after purposeful oxidation forfive weeks while one sample obtained from this same source becamerapidly bactericidal in as short as three weeks. Also, as shown in TableJ, both d- and l- isomers of limonene can be purposely oxidized tobecome a rapid bactericide. These data emphasize the need to test everybatch of purposely oxidized limonene after three to eight weekstreatment to ensure that the reference bactericidal endpoint has beenreached.

                  TABLE J                                                         ______________________________________                                        Antibacterial Activity Of Various Limonene Samples As                         Determined Against Staphylococcus aureus ATCC 25923                           During Purposeful Oxidation at Room Temperature.                                         Duration                                                                      of Pur-                                                                              Bactericidal Concentration                                             poseful                                                                              After Incubation For                                        Sample  Source   Oxidation                                                                              10 Mins                                                                              60 Min 24 Hours                              ______________________________________                                        d-limonene                                                                            Aldrich.sup.1                                                                          5 weeks  >1.sup.5                                                                             >1     >1                                    l-limonene                                                                            Aldrich.sup.1                                                                          5 weeks  >1     >1     >1                                    l-limonene                                                                            SCM.sup.2                                                                              3 weeks  >1     >1     >1                                    d-limonene                                                                            Intercit.sup.3                                                                         3 Weeks   0.1   0.005  0.0003                                l-limonene                                                                            SCM.sup.2                                                                              3 weeks   0.1   0.005  0.0006                                d-limonene                                                                            Aldrich.sup.1                                                                          3 weeks   0.005 0.0024 0.0003                                d-limonene                                                                            FCC.sup.4                                                                              6 weeks   0.16  0.06   0.0012                                ______________________________________                                         .sup.1 Aldrich, Milwaukee, Wisconsin                                          .sup.2 SCM Glidden Organics, Jacksonville, Florida                            .sup.3 Intercit, Inc., Safety Harbor, Florida                                 .sup.4 Florida Chemical Company, Inc., Lake Alfred, Florida              

The second method of producing RBE limonene is by continuously bubblingoxygen into limonene as outlined in Table K below. This process, likebubbling air through limonene produces a rapidly bactericidal oil andrequires 6 to 7 weeks treatment for the reference endpoint to beachieved.

                  TABLE K                                                         ______________________________________                                        Bactericidal Activity of d-limonene.sup.1 During Oxygenation As               Determined Against Staphylococcus aureus ATCC 25923                           Days d-Limonene                                                                            Bactericidal Concentration.sup.2                                 Oxidized.sup.3 at                                                                          Following Incubation For:                                        25° C. 15 Min    60 Min   24 Hours                                     ______________________________________                                         0 days       >1.sup.4  >1       >1                                            1 day        >1        >1       0.01                                          2 days       >1        >1       0.01                                          3 days       >1        >1       0.01                                          4 days       >1        >1       0.01                                          7 days       >1        >1       0.005                                         9 days       >1        >1       0.005                                        15 days        0.5      0.5      0.005                                        17 days        0.5      0.5      0.001                                        24 days       >1        0.16     0.005                                        36 days        0.5      0.10     0.0006                                       45 days        0.16     0.06     0.001                                        49 days        0.16     0.06     0.0003                                       53 days        0.3      0.06     0.0003                                       59 days        0.10     0.02     0.0003                                       66 days        0.16     0.01     0.0003                                       73 days        0.01     0.01     0.0003                                       ______________________________________                                         .sup.1 Fresh dlimonene (Intercit Inc., Safety Harbor, Florida lot 509801)     .sup.2 Results expressed as the highest dilution of limonene killing at       least 99.99% of the original inoculum.                                        .sup.3 Oxygen bubbled continuously into dlimonene at a rate of 3 liter pe     min at 25° C.                                                          .sup.4 Undiluted oil was not bactericidal.                               

EXAMPLE 2

In Example 2 the bactericidal and fungicidal activity of RBE d-limoneneand RBE l-limonene was determined against bacteria, yeasts, and fungi asoutlined in Tables L and M below.

                  TABLE L                                                         ______________________________________                                        BACTERICIDAL AND FUNGICIDAL ACTIVITY                                          OF RBE d-LIMONENE                                                             ______________________________________                                                          BACTERICIDAL                                                                  CONCENTRATION                                                                 AFTER INCUBATION                                                              FOR                                                         ORGANISM            10 Min  60 Min  24 Hours                                  ______________________________________                                        A. BACTERIA                                                                    1. Staphylococcus aureus,                                                                        0.3     0.06    0.0012                                       ATCC 25923                                                                  2. Staphylococcus aureus 3*                                                                      1.00    0.10    0.001                                      3. Staphylococcus aureus 27**                                                                    1.00    0.10    0.001                                      4. Staphylococcus aureus 37*                                                                     0.30    0.06    0.0025                                     5. Staphylococcus aureus 39**                                                                    0.30    0.06    0.0015                                     6. Staphylococcus aureus 52T                                                                     0.30    0.06    0.0012                                     7. Staphylococcus aureus 53T                                                                     0.30    0.06    0.0025                                     8. Staphylococcus aureus 54T                                                                     0.30    0.06    0.0025                                     9. Staphylococcus epidermidis 10                                                                 1.00    0.10    0.001                                     10. Sarcinia lutea  1.00    0.01    0.001                                     11. Streptococcus mitis 15 (alpha)                                                                0.01    0.01    0.001                                     12. Streptococcus pyogenes 1 (group A)                                                            0.01    0.01    0.01                                      13. Streptococcus salivarius (gamma)                                                              0.01    0.01    0.01                                      14. Streptococcus mutans                                                                          0.16    0.06    0.0012                                    15. Streptococcus faecalis 15                                                                     1.00    0.10    0.01                                      16. Lactobacillus casei M7                                                                        0.01    0.01    0.01                                      17. Listeria monocytogenes M9                                                                     0.01    0.01    0.01                                      18. Bacillus subtilis ATCC 6633                                                                   0.01    0.001   0.001                                     19. Escherichia coli 7                                                                            0.01    0.01    0.01                                      20. Klebsiella SP12 0.01    0.01    0.001                                     21. Salmonella 14 (paraB)                                                                         0.01    0.01    0.01                                      22. Shigella sonnei 8                                                                             0.01    0.001   0.001                                     23. Proteus mirabilis 2                                                                           0.01    0.01    0.01                                      24. Proteus vulgaris 127                                                                          0.10    0.01    0.01                                      25. Proteus rettgeri 106                                                                          0.10    0.01    0.10                                      26. Proteus Morganii 99                                                                           0.01    0.01    0.01                                      27. Providencia stuartii 97                                                                       0.01    0.01    0.01                                      28. Enterobacter cloacae 20                                                                       0.01    0.01    0.10                                      29. Citrobacter SP8 0.01    0.01    0.10                                      30. Pseudomonas aeruginosa 115                                                                    0.10    0.10    0.01                                      31. Pseudomonas mendocina 129                                                                     1.0     0.10    0.01                                      32. Neisseria gonorrhea 5                                                                         0.001   0.001   0.001                                     33. Neisseria gonorrhea (Pen R)*                                                                  0.001   0.001   0.001                                     34. Hemophilus influenzae (Amp R)**                                                               0.01    0.001   0.001                                     35. Hemophilus influenzae (Amp S)***                                                              0.001   0.001   0.001                                     36. Neisseria meningitidis                                                                        0.001   0.001   0.001                                     37. Xanthomonas campestris PV                                                                     0.0012  0.0003  0.0003                                       vesicatoria 1                                                              38. Xanthomonas campestris PV                                                                     0.0012  0.0003  0.0003                                       vesicatoria  2                                                             39. Xanthomonas (SP) *+ isolated                                                                  0.0025  0.0003  0.0003                                       from a Nebraska farmer                                                     40. Xanthomonas campestris PV citri                                                               0.005   0.0025  0.0006                                    41. Mycobacterium tuberculosis                                                                    0.001   0.001   0.0001                                       var hominis H37 RV                                                         42. Mycobacterium kansasii Tam 30                                                                 0.1     0.01    0.0001                                    43. Mycobacterium scrofulaceum                                                                    0.1     0.01    0.0001                                    44. Mycobacterium intracellulare                                                                  0.1     0.10    0.0001                                    45. Mycobacterium fortuitum                                                                       1.0     0.10    0.0001                                       ATCC 6841                                                                  B. YEASTS                                                                      1. Candida albicans                                                                              0.01    0.001   0.001                                      2. Sacchromyces SP 0.01    0.01    0.001                                     ______________________________________                                                    FUNGICIDAL CONCENTRATION                                                      AFTER INCUBATION FOR                                                            10 Min  60 Min  24 Hours                                                                             7-45 Days                                ______________________________________                                        C. FUNGI                                                                       1. Epidermophyton                   0.001                                       floccosum                                                                   2. Microsporum audouini             0.001                                     3. Microsporum canis                                                                       0.01    0.0025  0.0006                                           4. Trichophyton                     0.001                                       mentagrophytes                                                              5. Aureoblasidium                                                                          0.01    0.005   0.0012                                             pullulans OM 279C                                                           6. Cladosporium                                                                            0.01    0.005   0.0012                                             cladosporiodes OM 489                                                        7. Philopohora lignicola                                                                  0.10    0.01    0.0025                                             OM 5922                                                                    ______________________________________                                         *Penicillin resistant                                                         **Ampicillin resistant                                                        **Ampicillin sensitive                                                        *+ Species unidentified                                                  

                  TABLE M                                                         ______________________________________                                        BACTERICIDAL AND FUNGICIDAL ACTIVITY                                          OF RBE 1-LIMONENE                                                             ______________________________________                                                         BACTERICIDAL                                                                  CONCENTRATION                                                                 AFTER INCUBATION                                                              FOR                                                          ORGANISM           10 Min  60 Min   24 Hours                                  ______________________________________                                        A. BACTERIA                                                                    1. Staphylococcus aureus                                                                        0.5     0.06     0.0025                                       ATCC 25923                                                                  2. Staphylococcus aureus 37                                                                     0.5     0.40     0.005                                        (Pen R)*                                                                    3. Staphylococcus aureus 39                                                                     0.3     0.06     0.0025                                       (Meth R)**                                                                  4. Staphylococcus aureus 52T                                                                    0.5     0.10     0.01                                       5. Staphylococcus aureus 53T                                                                    0.5     0.10     0.0025                                     6. Staphylococcus aureus 54T                                                                    0.5     0.06     0.01                                       7. Staphylococcus epidermidis 10                                                                0.1     0.1      0.0025                                     8. Streptococcus mutans M42                                                                     0.3     0.16     0.005                                      9. Streptococcus faecalis 15                                                                    0.1     0.01     0.0025                                    10. Bacillus subtilis ATCC 6633                                                                  0.1     0.1      0.1                                       11. Escherichia coli 7                                                                           0.005   0.0025   0.0012                                    12. Salmonella 14 (Para B)                                                                       0.0025  0.0025   0.0006                                    13. Shigella sonnei 8                                                                            0.01    0.0025   0.0012                                    14. Pseudomonas aeruginosa 115                                                                   0.1     0.1      0.005                                     15. Neisseria meningitidis 40                                                                    0.0012  0.0006   0.00004                                   16. Xanthomonas campestris                                                                       0.0025  0.0012   0.0012                                       vesicatoria 1                                                              17. Xanthomonas campestris                                                                       0.0025  0.0012   0.0003                                       vesicatoria 2                                                              18. Xanthomonas SP isolated                                                                      0.0006  0.0006   0.0003                                       from a Nebraska farmer *+                                                  19. Xanthomonas campestris                                                                       0.005   0.0025   0.0006                                       PV citri                                                                   20. Mycobacterium tuberculosis                                                                   0.0005  0.000125 0.000125                                     var. hominis H37 RV                                                        21. Mycobacterium fortuitum                                                                      0.1     0.1      0.0006                                       ATCC 6841                                                                  B. YEAST                                                                       1. Candida albicans                                                                             0.0012  0.0012   0.0006                                    ______________________________________                                                          FUNGICIDAL                                                                    CONCENTRATION                                                                 AFTER INCUBATION                                                              FOR                                                                             10 Min  60 Min  24 Hours                                  ______________________________________                                        C. FUNGI                                                                       1. Microsporum canis                                                                             0.01    0.0025  0.0006                                     2. Aureobasidium pullulans OM 279C                                                               0.1     0.005   0.0012                                     3. Cladosporium cladosporiodes                                                                   0.01    0.005   0.0012                                       OM 489                                                                      4. Phialophora lignicola OM 5922                                                                 0.1     0.01    0.005                                     ______________________________________                                         *Penicillin resistant                                                         *Methicillin resistant                                                        *+ Unidentified species                                                  

The standard assay used to test the bactericidal activity of the d and lisomers limonene which had been oxidized until they reached thereference bactericidal endpoint was as follows: various dilutions of thedesired limonene were separately prepared in an appropriate broth mediumfor the test strain. An inoculum of 10⁶ colony-forming units (CFU)/mlwas used. Each test was incubated at the proper temperature for eachorganism and subcultured (0.01 ml) at 10 minutes, 60 minutes and 24hours onto agar media free of limonene.

Results were expressed as the bactericidal concentration, i.e., thelowest concentration of RBE limonene (ml limonene/total ml of test)killing at least 99.99% of the bacterial inoculum.

The activity of RBE limonene was evaluated against Mycobacteria usingundiluted oil, and oil diluted up to 1:8000 in Proskauer Beck liquidmedium. Each test was inoculated with 10⁶ CFU/ml of Mycobacteria andincubated at 37 degrees C in 7% CO₂ in air. At various time intervals,each test was shaken vigorously and a 0.01 ml aliquot removed. This wassubcultured onto Dubos oleic acid-albumin agar plates to determine thenumber of viable Mycobacteria remaining. Each test was sampled in thisfashion after incubation for 10 minutes, 60 minutes, 24 hours, 1, 2, 3,and 4 weeks. The bacterial concentration was defined as the lowestconcentration of oil killing at least 99.99% of the original inoculum.

The standard assay used to test certain fungi (dermatophytes) was asfollows: RBE d-limonene and RBE 1-limonene were tested in air onsabouraud dextrose agar slants supplemented with 0.05% yeast extract.Each of the two compounds was individually added to different samples ofmolten agar in dilutions of 1:10, 1:15, 1:100, 1:1000 and then mixedvigorously after which the agar was allowed to solidify. The inoculumwas prepared as follows: 10 ml of saline (0.85%) and four drops of tween80 were added to sabouraud dextrose agar slants containing the fungusgrown for 14 days at room temperature. After vigorous shaking, theculture was filtered through sterile gauze and adjusted to the densityof a MacFarland Standard No. 1 with 0.85% saline. The limonenecontaining slants were inoculated with 0.1 ml of this adjustedsuspension, incubated at room temperature and checked for growth dailyfor 43 days. After 7 days, confluent growth of each fungus was observedon limonene-free control slants. The fungicidal concentration of RBElimonene was defined as the lowest concentration preventing growth onthe slants for 43 days.

The test conditions used to assay the antimicrobial activity of RBElimonene are listed in Table N which follows.

                  TABLE N                                                         ______________________________________                                                               SUB-       INCU-                                                              CULTURE    BATION                                                   BROTH     AGAR       CONDI-                                      ORGANISM     MEDIUM    MEDIUM     TIONS                                       ______________________________________                                         1. Staphylococcus,                                                                        Mueller-  5% sheep   air @ 37° C.                            Sarcina, Bacillus,                                                                      Hinton    blood                                                     Enterobacteriacea,                                                            and Pseudomonas                                                             2. Streptococcus, Lac-                                                                    Todd-     5% sheep   10% CO.sub.2 in                                tobacillus, Listeria                                                                    Hewitt    blood      air @ 37° C.                          3. Neisseria                                                                              Mueller-  5% sheep   10% CO.sub.2 in                                meningitidis                                                                            Hinton    blood      air @ 37° C.                          4. Neisseria gonorrhea                                                                    Eugon     chocolate  10% CO.sub.2 in                                                               air @ 37° C.                          5. Hemophilus                                                                             Levinthal chocolate  10% CO.sub.2 in                                influenza                      air @ 37° C.                          6. Yeast    Sabouraud 5% sheep   air @ 37° C.                                      dextrose  blood                                                   7. Xanthomonas                                                                            Nutrient  blood agar air @ 25° C.                            campestris                                                                    vesicatoria                                                                   & citri                                                                     8. Xanthomonas                                                                            Mueller-  blood agar air @ 37° C.                            species isolated                                                                        Hinton                                                              from Nebraska                                                                 farmer (species                                                               not identified)                                                             9. Mycobacteria                                                                           Proskauer Dubos-oleic                                                                              7% CO.sub.2 @                                            Beck      acid-albumin                                                                             37° C.                                                      agar                                                   10. Fungi    Sabourauds                                                                    dextrose                                                                      with 0.05%                                                                    yeast                                                                         extract                                                          ______________________________________                                    

EXAMPLE 3

In example 3, formulations, containing RBE limonene are listed.

RBE d-limonene and RBE 1-limonene are prepared in the followingformulations including liquids, gels, soaps, paints, pastes, creams,ointments, suppositories, tampons, aerosols, and emulsions. Whenbacteria, fungi or yeasts are treated with formulations that contain RBEd-limonene or RBE 1-limonene, the formulations kill or prevent thegrowth of bacteria, yeasts, and fungi.

    ______________________________________                                                      % OF                                                            CHEMICAL      TOTAL    RANGE      ACTION                                      ______________________________________                                        A. LIQUIDS                                                                    1. SOLUTIONS OR SPRAYS                                                        a. RBE d-Limonene                                                                           6.0%     0.1-50%    bactericide                                  Corn Oil     94.0%      50-99.9% diluent                                                   100.0%                                                          b. RBE 1-Limonene                                                                           1.0%     0.1-50%    fungicide                                    Ethyl Alcohol                                                                              99.0%      50-99.9% diluent                                                   100.0%                                                          2. MOUTHWASH                                                                  a. RBE d-limonene                                                                           50.0%    0.1-50%    antiyeast                                    Flavor       2.0%     1-5%       flavor                                       Ethyl Alcohol                                                                              48.0%      45-98.9% diluent                                                   100.0%                                                          B. DENTIFRICE                                                                 1. LIQUID                                                                     Liquid soap concentrate                                                                     5.0%      2-10%     surfactant                                  Saccharin     0.2%     0.1-1.0%   flavor                                      Clove Oil     1.0%     0.5-3.0%   flavor                                      Cinnamon Oil  0.5%     0.5-3.0%   flavor                                      Peppermint Oil                                                                              0.5%     0.5-3.0%   flavor                                      Ethyl Alcohol 42.6%    29.5-95.3% diluent                                     Color         0.2%     0.1-0.5%   color                                       RBE 1-limonene                                                                              50.0%     1-50%     fungicide                                                 100.0%                                                          2. GEL                                                                        Sodium monofluoro-                                                                          0.8%     0.5-1.5%   antiplaque                                  phosphate                                                                     RBE d-limonene                                                                              50.0%     1-50%     antibacterial                               Hydrated silica xerogel                                                                     10.0%     8-15%     abrasive                                    Hydrated thickening                                                                         8.5%      5-10%     binder                                      silica                                                                        Sorbitol 70% solution                                                                       18.8%       5-73.3% humectant                                   Polyethylene glycol 32                                                                      5.0%     3-7%       bodying agent                               Sodium lauryl sulfate                                                                       1.5%     1-2%       surfactant                                  Carboxymethyl cellulose                                                                     1.0%     0.5-2%     binder                                      gum                                                                           S D alcohol   1.0%     0.5-2%     stabilizer                                  Flavor        3.0%     2-4%       flavor                                      Saccharin     0.2%     0.1-0.5%   flavor                                      F D & C Green #3                                                                            0.1%     0.1-0.5%   color                                       F D & C Yellow #10                                                                          0.1%     0.1-0.5%   color                                                     100.0%                                                          3. PASTE                                                                      Sodium monofluoro-                                                                          0.8%     0.5-1.5%   antiplaque                                  phosphate                                                                     RBE 1-limonene                                                                              50.0%     1-50%     fungicide                                   Dicalcium phosphate                                                                         22.0%    20.4-30%   abrasive                                    dihydrate                                                                     Water         16.0%    11.1-69.5% diluent                                     Glycerine     5.1%      4.5-12.5% bodying agent                               Flavor        2.0%     2-3%       flavor                                      Sodium lauryl sulfate                                                                       1.5      1-2%       surfactant                                  Carboxymethyl cellulose                                                                     1.4%     0.5-2.0%   binder                                      gum                                                                           Tetrasodium pyrophos-                                                                       1.0%     0.5-2.0%   binder                                      phate                                                                         Sodium saccharin                                                                            0.2%     0.1-0.5%   flavor                                                    100.0%                                                          C. OINTMENTS & SUPPOSITORIES WITH AND WITHOUT                                 HYDROCORTISONE                                                                1. OINTMENT WITH HYDROCORTISONE                                               RBE d-limonene                                                                              1.0%      0.1-15.0% bactericide                                 Polyethylene glycol                                                                         59.0%    48.5-59.7% bodying agent                               3350                              & emulsifier                                Polyethylene glycol                                                                         39.0%    31.5-39.7% bodying agent                               400                               & emulsifier                                Hydrocortisone                                                                              1.0%     0.5- 5.0%  anti-                                                     100.0%              inflammatory                                2. OINTMENT WITHOUT HYDROCORTISONE                                            RBE 1-limonene                                                                              1.0%      0.1-15.0% fungicide                                   Polyethylene glycol                                                                         59.5%     51.0-59.95%                                                                             bodying agent                               3350                              & emulsifier                                Polyethylene glycol                                                                         39.5%     34.0-39.95%                                                                             bodying agent                               400           100.0%              & emulsifier                                3. SUPPOSITORY WITHOUT HYDROCORTISONE                                         RBE 1-limonene                                                                              6.0%     0.1-15%    bactericide                                 Polyethylene glycol                                                                         56.5%     51.0-59.95%                                                                             bodying agent                               1000                              & emulsifier                                Polyethylene glycol                                                                         37.5%     34.0-39.95%                                                                             bodying agent                               3350          100.0%              & emulsifier                                4. SUPPOSITORY WITH HYDROCORTISONE                                            RBE d-limonene                                                                              1.0%     0.1-15%    antiyeast                                   Polyethylene glycol                                                                         74.0%    60.0-75.2% bodying agent                               1000                              & emulsifier                                Polyethylene glycol                                                                         24.0%    20.0-24.2% bodying agent                               3350                              & emulsifier                                Hydrocortisone                                                                              1.0%     0.5-5.0%   anti-                                                     100.0%              inflammatory                                D. CREAMS WITHOUT HYDROCORTISONE                                              1. RBE 1-limonene                                                                           6.0%      0.1-15.0% bactericide                                  Cetyl alcohol                                                                              15.0%    12.0-18.0% thickener                                    Arlacel 165**                                                                              5.0%     3.5-7.5%   emulsifier                                   Sorbitol 70% solution                                                                      5.0%     3.5-8.0%   humectant                                    Water        69.0%    51.5-80.9% diluent                                                   100.0%                                                          2. RBE 1-limonene                                                                           1.0%      0.1-15.0% fungicide                                    Spermaceti wax                                                                             12.5%    10.0-15.0% thickener                                    Sorbitan monostea-                                                                         10.0%     7.5-12.5% emulsifier                                   rate* Polyethylene 20                                                         Sorbitan monostea-                                                                         6.0%     4.0-8.0%   emulsifier                                   rate*                                                                         Water        70.5%    49.5-78.4% diluent                                                   100.0%                                                          E. CREAMS WITH HYDROCORTISONE                                                 1. RBE d-limonene                                                                           1.0%      0.1-15.0% fungicide                                    Cetyl alcohol                                                                              15.0     12.0-18.0% thickener                                    Arlacel 165**                                                                              5.0      3.5-7.5%   emulsifier                                   Sorbitol 70% solution                                                                      5.0%     3.5-8.0%   humectant                                    Hydrocortisone                                                                             1.0%     0.5-5.0%   anti-                                                                         inflammatory                                 Water        73.0%    46.5-80.4% diluent                                                   100.0%                                                          F. TAMPONS                                                                    1. RBE d-limonene                                                                            8%       1-15%     bactericide                                 2 cc 2 Gm                                                                      Tampon 23 Gm     92%      85-99%   reservoir for                                               100%              fungicide                                 G. AEROSOLS WITHOUT HYDROCORTISONE                                            1. RBE 1-limonene                                                                           6.0%     0.5-50%    bactericide                                  Ethyl alcohol                                                                              94.0%      50-99.5% diluent                                                   100.0%                                                           Pressurized nitrogen                                                          propellant at 100-125                                                         psig                                                                         2. RBE d-limonene                                                                           10.0%     0.5-50.0% fungicide                                    Soybean Oil  90.0%    50.0-99.5% diluent                                                   100.0%                                                           Pressurized nitrogen                                                          propellant at 100-125                                                         psig                                                                         H. AEROSOL WITH HYDROCORTISONE                                                1. RBE 1-limonene                                                                           6.0%     0.5-50%    bactericide                                  Soybean oil  93.0%      45-99.0% diluent                                      Hydrocortisone                                                                             1.0%     0.5-5.0%   anti-                                                     100.0%              inflammatory                                 Pressurized nitrogen                                                          propellant at 100-125                                                         psig                                                                         I. OIL IN WATER EMULSION                                                      1. RBE d-limonene                                                                           0.1%     0.1-50%    fungicide                                   2. Corn oil   10.0%    10-15%     oil                                          Arlacel 40** 2.0%     1-3%       emulsifier                                   Tween 40     3.0%     2-4%       emulsifier                                  3. Water      84.9%      28-86.9% diluent                                                   100.0%                                                          Heat 2 to 70° C. Heat 3 to 72° C. Add 3 to 2 with               continuous                                                                    agitation. When 3 and 2 cool to 40° C., add 1 with continuous          agitation until room temperature is reached.                                  J. OIL IN WATER EMULSION WITH SOAP                                            (FUNGICIDAL SOAP)                                                             1. RBE d-limonene                                                                           10.0%    0.1-25%    bactericide                                 2. Corn oil   21.0%    20.0-40.0% oil                                          Arlacel 40** 2.0%     1.0-3.0%   emulsifier                                   Tween 40     3.0%     2.0-4.0%   emulsifier                                   Liquid soap concen-                                                                        3.5%     2.5- 5.0%  surfactant                                   trate                                                                        3. Water      60.5%      23-74.4% diluent                                                   100.0%                                                          Heat 2 to 70° C. Heat 3 to 72° C. Add 3 to 2 with               continuous                                                                    agitation. When 3 and 2 cool to 40° C., add 1 with continuous          agitation until room temperature is reached.                                  K. WATER IN OIL EMULSION                                                      1. RBE 1-limonene                                                                           1.0      0.1-25%    fungicide                                   2. Arlacel 186**                                                                            3.0      2.0-4.0%   emulsifier                                   Soybean oil  15.0%    10.0-25.0% oil                                          Ceresin wax  0.5%     0.3-0.6%   thickener                                    Beeswax      0.5%     0.3-0.6%   thickener                                    Tween 80     0.5%     0.3-0.6    emulsifier                                  3. Water      79.5%    44.2-87.0% diluent                                                   100.0%                                                          Heat 2 to 70° C. Heat 3 to 72° C. Add 3 to 2 with               continuous                                                                    agitation. When 3 and 2 cool to 40° C., add 1 with continuous          agitation until room temperature is reached.                                  L. PAINT                                                                      1. ENAMEL                                                                     RBE d-limonene                                                                              1.00%     1-10%     fungicide                                   Titanium dioxide                                                                            14.91%   12-16%     pigment                                     Calcium carbonate                                                                           29.83%   25-35%     pigment                                     Silicate      4.81     3-6%       pigment                                     Soya alkyd resin                                                                            25.72%   22-28%     pigment                                                                       (binder)                                    Mineral spirits                                                                             23.73%    5-37%     solvent                                                   100.00%             (thinner)                                   2. LATEX                                                                      RBE 1-limonene                                                                              1.00%     1-10%     fungicide                                   Titanium dioxide                                                                            10.76%    8-12%     pigment                                     Silicate      12.91%   10-16%     pigment                                     Calcium carbonate                                                                           20.91%   15-25%     pigment                                     Vinyl acrylic resin                                                                         12.22%   10-16%     vehicle                                     solids                            (binder)                                    Glycol        8.30%     6-10%     solvent                                                                       (thinner)                                   Water         34.00%   12-50%     solvent                                                   100.00%             (thinner)                                   ______________________________________                                         *Croda, Inc., 51 Madison Ave., New York, New York 10010                       **Glycerol monostearate and polyoxyethylene stearate ICI of America           (Formerly Atlas Chemical Industries), Wilmington, Delaware 19899         

While only certain preferred embodiments of this invention have beenshown and described by way of illustration, many modifications willoccur to those skilled in the art and it is, therefore, desired that itbe understood that it is intended herein, to cover all suchmodifications that fall within the true spirit and scope of thisinvention.

We claim:
 1. Reference bactericidal and fungicidal end point limonene asa composition of matter that is bactericidal and fungicidal inbactericidal and fungicidal concentrations comprising oxidized limonenethat is capable of killing at least 99.99% of the bacteria in a suitableculture medium at a concentration of 0.06 ml. per ml. of the culturemedium inoculated with 10⁶ colony forming units of staphylococcus aureusATCC 25923 after being inoculated at 37° in air for sixty minutes. 2.The composition of matter of claim 1 wherein said oxidized limonene isprepared by bubbling air or oxygen into limonene.
 3. The composition ofmatter of claim 2 wherein said bubbling is accomplished at roomtemperature for about 3-8 weeks.